Study On The Metabolic Mechanism Of Soy Sauce Flavor By Knockout PckA And MsmE In Bacillus Subtilis

As a traditional fermented flavor in China,Soy sauce is favored because of its unique flavor.Now,there are many gaps in the mechanism of producing the flavor of Douchi fermented by Bacillus subtilis.By screening and analyzing the transcriptional sequencing of the Bacilus subtilis BJ3-2,at medium-high temperature(45?)which produce soy sauce and medium-low temperature(37?)which produce Douchi flavor,several differentially significant genes were obtained.Based on the bioinformatics analysis of different genes,phosphoenolpyruvate carboxykinase(PCKA),raffinose/stachyose/melibiose transport system substrate-binding protein(MSME)were selected as candidate genes to study their functions in the production of soy sauce by Bacillus subtilis.The main research contents and results are as follows:1.The metabolic pathway of candidate genes pck A,msm E and flavor metabolism of soy sauce were mapped by bioinformatics analysis.pck A is involved in the metabolism of tricarboxylic acid cycle,converting oxaloacetic acid into phosphoenolpyruvate,phosphoenolpyruvate is converted into pyruvate by pyruvate kinase.Pyruvate is converted into acetoin by enzyme,acetoin reacts with ammonium to form the soy sauce flavor substance tetramethylpyrazine;msm E is involved in the transport of oligosaccharides and controls the transport of oligosaccharides inside and outside the cell.Glucose is decomposed into pyruvate through glycolysis.Pyruvate generates acetoin under the action of enzyme,and then reacts with ammonium to form the soy sauce flavor substance tetramethylpyrazine.2.The expression of pck A and msm E of Bacilus subtilis BJ3-2 in 37?and 45?was determined by q PCR.16 S r RNA was selected as the internal reference gene.The results showed that the expression of pck A and msm E at 45?was 0.57 and 1.38 fold than that at 37?,respectively.Its consistent with the result of transcriptome sequencing.3.pck A and msm E homologous recombination double exchange knockout vector p UC18+HLarm+cat+HRarm were contructed.Using p UC18 as the initial vector,the left arm (HLarm),the right arm(HRarm)and the Chloramphenicol Acetyltransferase(cat)were successively linked by Eco R I,Sac I,Bam H I and Pst I.Through test of colony PCR,plasmid PCR,double enzyme digestion and sequencing,the homologous recombination knockout vector p UC18+HLarm+cat+HRarm was finally obtained.4.The double exchange knockout plasmid was transformed into the original strain of Bacillus subtilis BJ3-2,and the gene knockout strains BJ3-2 ? pck A and BJ3-2 ? msm E were successfully obtained.5.Using BJ3-2?pck A and BJ3-2?msm E to ferment Douchi,the volatile flavor compounds in Douchi were detected by GC-MS and evaluated by sensory evaluation.Compared with Douchi fermented by BJ3-2,Douchi fermented by BJ3-2?pck A the soy sauce is rich in flavor and thick after fermentation,there are a lot of viscold between the beans;Douchi fermented by BJ3-2?msm E the Douchi is dark brown in color,thick in texture,weak in soy sauce flavor and there are a lot of viscold between the beans.The results of GC-MS showed that compared with Douchi fermented by BJ3-2,Douchi fermented by BJ3-2?pck A the content of acetoin decreased from8.618% to 7.721%,the content of tetramethylpyrazine increased from 12.444% to 15.394%,.Meanwhile,Douchi fermented by BJ3-2?msm E the content of acetoin increased from 8.618% to28.122%,the content of tetramethylpyrazine decreased from 12.444% to 9.124%.At last,it is concluded that pck A can regulate the formation of sauce flavor negatively,and the formation of soy sauce flavor was positively regulated by msm E.The study detected the volatile flavor substances by GC-MS and found that medium-high temperature can make Bacillus subtilis BJ3-2 produce soy sauce flavor;By successfully knocking out pck A and msm E.It was found that pck A was a negative regulatory gene of soy sauce flavor,which affected the metabolism of pyruvate by regulating the production of phosphoenolpyruvate in the cycle of tricarboxylic acid;msm E is a positive regulatory gene of soy sauce flavor,which regulates the metabolism of pyruvate in glycolysis by regulating the transport of oligosaccharides inside and outside cells;After knockout msm E,the soy sauce flavor of Douchi decreased and the browning degree of Douchi deepened,which indicated that soy sauce flavor of Douchi was not positively correlated with the browning degree.